Bacteriophage lambda (phage .lambda.) is known as a convenient vector with which to isolate recombinant DNA sequences. In general, a foreign DNA molecule is inserted into the phage .lambda. genome and propagated along with the native phage .lambda. sequences. In order to propogate the phage, a system must be developed to "package" the phage.
The first reports of in vitro phage .lambda. DNA packaging systems were described in Sternberg, N., Triemeier, D., and Enquist, L., Gene 1:255-280, 1977 and Hohn, B. and Murray, K., Proc. Natl. Acad. Sci. USA 74:3259-3263, 1977. These systems were not high efficiency (greater than 1.times.10.sup.8 pfu/.mu.g wild type .lambda. DNA) packaging systems.
Some early .lambda. packaging systems relied on a combination of extracts to achieve the packaging function. However, it is more convenient to use a single extract. A single extract packaging system was first described in Rosenberg, S.M., Stahl, M.M., Kobayashi, I., and Stahl, F., Gene. 38:165-175, 1986. This paper described a single bacterial strain useful for in vitro packaging of .lambda. DNA. The strain was an E. coli C strain that carried a prophage that was deleted for the nicking site of action of terminase, cosN. (The terminase multimer in phage .lambda. is made up of two proteins that provide a number of functions in .lambda. DNA packaging, such as DNA recognition, prohead binding, cos site cleavage and probably the entry of the DNA into the prohead.) This cos deletion prevents the packaging of the endogenous prophage. Therefore, packaging extracts made from this lysogen produce no background of packaged endogenous DNA. The C strain of E. coli lacks any known DNA restriction system, thereby eliminating the possibility that DNA packaged in this system will be biased by restriction of the DNA. The extract described by Rosenberg, et al. is prepared by concentration of the induced lysogen followed by freezing. The extract was stable only when stored at -70.degree. C.
The use of a plasmid that overexpresses the terminase genes as a way of enhancing packaging extracts was first described in Murialdo, H., Davidson, A., Chow, S., and Gold, M., Nucl. Acids Res. 15:119-140, 1987 and Chow, S., Daub, E., and Murialdo, H., Gene. 60:277-289, 1987. These references describe the use of packaging extracts produced from two E. coli K12 lysogens supplemented by an extract of another E. coli K12 strain carrying a plasmid that expresses the terminase subunits. This system utilizes sonicated extracts of two different bacterial strains and a freeze/thaw lysate of a third strain. The packaging extracts are unstable at storage temperatures above -70.degree. C.
Current procedures for making .lambda. DNA packaging extracts from a single lysogen and from the two lysogen system are included in Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989. 2.95-2.107. The procedure for making single lysogen extracts that is provided does not use sonication and is said to produce extracts with an efficiency of 0.2.times.10.sup.8 -1.times.10.sup.8 pfu/.mu.g wild type .lambda. DNA. The references describe storage at -70.degree. C.
.lambda. DNA packaging extracts are now commercially available from a number of companies at efficiencies of 1.times.10.sup.8 -20.times.10.sup.8 pfu/.mu.g wild type .lambda. DNA. All of the presently available packaging extracts are unstable during storage at temperatures above -70.degree. C.
Therefore, a single-extract in vitro phage .lambda. packaging system that is stable during storage at ambient temperature is needed.